Combining different 'omics' technologies to map and validate protein-protein interactions in humans.

The mapping of protein-protein interactions is key to understanding biological processes. Many technologies have been reported to map interactions and these have been systematically applied in yeast. To date, the number of reported yeast protein interactions that have been truly validated by at least one other approach is low. The mapping of human protein interaction networks is even more complicated. Thus, it is unreasonable to try to map the human interactome; instead, interaction mapping in human cell lines should be focused along the lines of diseases or changes that can be associated with specific cells. In this paper, an approach for combining different 'omics' technologies to achieve efficient mapping and validation of protein interactions in human cell lines is presented.     

Failure tolerance of motif structure in biological networks

Complex networks serve as generic models for many biological systems that have been shown to share a number of common structural properties such as power-law degree distribution and small-worldness. Real-world networks are composed of building blocks called motifs that are indeed specific subgraphs of (usually) small number of nodes. Network motifs are important in the functionality of complex networks, and the role of some motifs such as feed-forward loop in many biological networks has been heavily studied. On the other hand, many biological networks have shown some degrees of robustness in terms of their efficiency and connectedness against failures in their components. In this paper we investigated how random and systematic failures in the edges of biological networks influenced their motif structure. We considered two biological networks, namely, protein structure network and human brain functional network. Furthermore, we considered random failures as well as systematic failures based on different strategies for choosing candidate edges for removal. Failure in the edges tipping to high degree nodes had the most destructive role in the motif structure of the networks by decreasing their significance level, while removing edges that were connected to nodes with high values of betweenness centrality had the least effect on the significance profiles. In some cases, the latter caused increase in the significance levels of the motifs.     

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.     

On the number of experiments required to find the causal structure of complex systems

The need to capture the complexity of biological systems in a simpler formalism is the underlying impetus of biological sciences. Understanding the function of many biological complex systems, such as genetic networks or molecular signalling pathways, requires precise identification of the interactions between their individual components. A number of questions in the study of complex systems are then important-in particular, what can be inferred about the interactions in a complex system from an arbitrary set of experiments, and, what is the minimum number of experiments required to characterize the system? This paper shows that the problem of finding the minimal causal structure of a system based on a set of observations is computationally intractable for even moderately sized systems (it is NP-hard), but a reasonable approximation can be found in a relatively short (polynomial) time. Next, it is shown that the number of experiments required to characterize a complex system grows exponentially with the upper bound on the number of immediate upstream influences of each element, but only logarithmically with the number of elements in the system. This makes it possible to study biological systems with extremely large number of interacting elements and relatively sparse interconnections, such as gene regulatory and cell signalling networks. Finally, the construction of a randomized experimental sequence which achieves this bound is discussed.     

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are “scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.     

Stochastic simulations of minimal self-reproducing cellular systems

This paper is a theoretical attempt to gain insight into the problem of how self-assembling vesicles (closed bilayer structures) could progressively turn into minimal self-producing and self-reproducing cells, i.e. into interesting candidates for (proto)biological systems. With this aim, we make use of a recently developed object-oriented platform to carry out stochastic simulations of chemical reaction networks that take place in dynamic cellular compartments. We apply this new tool to study the behaviour of different minimal cell models, making realistic assumptions about the physico-chemical processes and conditions involved (e.g. thermodynamic equilibrium/non-equilibrium, variable volume-to-surface relationship, osmotic pressure, solute diffusion across the membrane due to concentration gradients, buffering effect). The new programming platform has been designed to analyse not only how a single protometabolic cell could maintain itself, grow or divide, but also how a collection of these cells could 'evolve' as a result of their mutual interactions in a common environment.     

The intracellular antibody capture technology: towards the high-throughput selection of functional intracellular antibodies for target validation

Several approaches have been developed over the past decade to study the complex interactions that occur in biological system. The ability to carry out a comprehensive genetic analysis of an organism becomes more limited and difficult as the complexity of the organism increases because complex organisms are likely to have not only more genes than simple organisms but also more elaborate networks of interactions among those genes. The development of technologies to systematically disrupt protein networks at the genomic scale would greatly accelerate the comprehensive understanding of the cell as molecular machinery. Intracellular antibodies (intrabodies) can be targeted to different intracellular compartments to specifically interfere with function of selected intracellular gene products in mammalian cells. This technique should prove important for studies of mammalian cells, where genetic approaches are more difficult. In the context of large-scale protein interaction mapping projects, intracellular antibodies (ICAbs) promise to be an important tool to knocking out protein function inside the cell. In this context, however, the need for speed and high throughput requires the development of simple and robust methods to derive antibodies which function within cells, without the need for optimization of each individual ICAb. The successful inhibition of biological processes by intrabodies has been demonstrated in a number of different cells. The performance of antibodies that are intracellularly expressed is, however, somewhat unpredictable, because the reducing environment of the cell cytoplasm in which they are forced to work prevents some antibodies, but not others, to fold properly. For this reason, we have developed an in vivo selection procedure named Intracellular Antibody Capture Technology (IACT) that allows the isolation of functional intrabodies. The IAC technology has been used for the rapid identification of antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by the selected intracellular antibodies. Several optimizations of the IAC technology for protein knock-out have been developed so far. This system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in different cell lines.     

Toward the assembly of a minimal divisome

The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.     

Synthetic biology of minimal systems

“Synthetic biology” is a concept that has developed together with, or slightly after, “systems biology”. But while systems biology aims at the full understanding of large systems by integrating more and more details into their models, synthetic biology phrases different questions, namely: what particular biological function could be obtained with a certain known subsystem of reduced complexity; can this function be manipulated or engineered in artificial environments or genetically modified organisms; and if so, how? The most prominent representation of synthetic biology has so far been microbial engineering by recombinant DNA technology, employing modular concepts known from information technology. However, there are an increasing number of biophysical groups who follow similar strategies of dissecting cellular processes and networks, trying to identify functional minimal modules that could then be combined in a bottom-up approach towards biology. These modules are so far not as particularly defined by their impact on DNA processing, but rather influenced by core fields of biophysics, such as cell mechanics and membrane dynamics. This review will give an overview of some classical and some quite new biophysical strategies for constructing minimal systems of certain cellular modules. We will show that with recent advances in understanding of cytoskeletal and membrane elements, the time might have come to experimentally challenge the concept of a minimal cell.     

JNK Signalling Controls Remodelling of the Segment Boundary through Cell Reprogramming during Drosophila Morphogenesis

Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.     

Mechanisms for quality control of misfolded transmembrane proteins

To prevent the accumulation of misfolded and aggregated proteins, the cell has developed a complex network of cellular quality control (QC) systems to recognize misfolded proteins and facilitate their refolding or degradation. The cell faces numerous obstacles when performing quality control on transmembrane proteins. Transmembrane proteins have domains on both sides of a membrane and QC systems in distinct compartments must coordinate to monitor the folding status of the protein. Additionally, transmembrane domains can have very complex organization and QC systems must be able to monitor the assembly of transmembrane domains in the membrane. In this review, we will discuss the QC systems involved in repair and degradation of misfolded transmembrane proteins. Also, we will elaborate on the factors that recognize folding defects of transmembrane domains and what happens when misfolded transmembrane proteins escape QC and aggregate. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Compartments and appendage development in Drosophila

The appendages of Drosophila develop from the imaginal discs. During the extensive growth of these discs cell lineages are for the most part unfixed, suggesting a strong role for cell-cell interactions in controlling the final pattern of differentiation. However, during early and middle stages of development, discs are subdivided by strict lineage restrictions into a small number of spatially distinct compartments. These compartments appear to be maintained by stably inheriting states of gene expression; the compartmentspecific expression of two such ‘selector’ - like genes, engrailed and apterous, are critical for anterior-posterior and dorso-ventral compartmentalization, respectively. Recent work suggests that one purpose of compartmentalization is to establish regions of specialized cells near compartment boundaries via intercompartmental induction, using molecules like the hedgehog protein. Thus, compartments can act as organizing centers for patterning within compartments. Evidence for non-compartmental patterning mechanisms will also be discussed.     

Gonadal cell lineages of the nematode Panagrellus redivivus and implications for evolution by the modification of cell lineage

To explore the nature of cell lineage modifications that have occurred during evolution, the gonadal cell lineages of the nematode Panagrellus redivivus have been determined and compared to the known gonadal lineages of Caenorhabditis elegans (J. Kimble and D. Hirsh, 1979, Develop. Biol.70, 396–417). Essentially invariant lineages generate the 143 somatic cells of the male gonad and at least 326 somatic cells of the female gonad of P. redivivus. The basic program of gonadogenesis is strikingly similar among both sexes of both species. For example, the early division patterns of the somatic gonad precursors Z1 and Z4 are almost identical. Later division patterns are more divergent and, in a few cases, generate structures that are species specific. In general, similar cell types are produced after similar patterns of cell divisions. Differences among the Z1 and Z4 cell lineages appear to reflect phylogenetic modifications of a common developmental program. The nature of these differences suggests that the evolution of cell lineages involves four distinct classes of alterations: switches in the fate of a cell to that normally associated with another cell; reversals in the polarity of the lineage generated by a blast cell; alterations in the number of rounds of cell division; and an “altered segregation” of developmental potential, so that a potential normally associated with one cell instead becomes associated with its sister. A number of cell deaths occur during gonadogenesis in P. redivivus. The death of Z4.pp, a cell that controls the development of the posterior ovary in C. elegans, probably prevents the development of a posterior ovary in P. redivivus and hence is responsible for the gross difference in the morphologies of the gonads of the P. redivivus female and the C. elegans hermaphrodite. As exemplified by the death of Z4.pp, an alteration in the fate of a “regulatory cell” could facilitate rapid and/or discontinuous evolutionary change.     

Dual targeting of plastid division protein FtsZ to chloroplasts and the cytoplasm

FtsZ is a filament-forming protein that assembles into a ring at the division site of prokaryotic cells. As FtsZ and tubulin share several biochemical and structural similarities, FtsZ is regarded as the ancestor of tubulin. Chloroplasts--the descendants of endosymbiotic bacteria within plant cells--also harbour FtsZ. In contrast to eubacteria, plants have several different FtsZ isoforms. So far, these isoforms have only been implicated with filamentous structures, rings and networks, inside chloroplasts. Here, we demonstrate that a novel FtsZ isoform in the moss Physcomitrella patens is located not only in chloroplasts but also in the cytoplasm, assembling into rings in both cell compartments. These findings comprise the first report on cytosolic localization of a eukaryotic FtsZ isoform, and indicate that this protein might connect cell and organelle division at least in moss.